The resident gut microbiome is intimately involved in host physiology and homeostasis. How these microbes establish and maintain their population in the complex and frequently stressed environment of the gastrointestinal (GI) tract is an important question. The genus Bacteroides is a major bacterial component of the GI tract and is rarely found elsewhere. Genomic analyses and genetic studies revealed that these bacteria have extraordinary ability for successful colonization in the mammalian intestine. Bacteroides spp. also have in their membranes a distinct lipid component-sphingolipids, which are otherwise thought to only be present in the membranes of eukaryotic cells. Eukaryotic sphingolipids species have been well-established as key signal transduction molecules in response to various cell stresses. The biological outcome of the signaling is profound and diverse, ranging from apoptosis to angiogenesis. Cholesterol is also a key coordinator in stress responses by helping to form the signaling platform in the eukaryotic membranes. It is interesting that as inhabitants of mammalian intestine, Bacteroides spp. have easy access to abundant cholesterol from dietary intake of the host or from biliary secretions. Therefore these bacteria are equipped with the basic components required for eukaryotic-like sphingolipid-mediated signaling. Preliminary studies show that the model organism Bacteroides fragilis has significantly improved survival against 4 different stresses in a sphingolipids/cholesterol-dependent manner. In addition, it is demonstrated by atomic force microscopy that the addition of cholesterol to purified B. fragilis membrane lipids considerably changes the physical organization of these lipids with the appearance of large sphingolipid- enriched domains. When stress is applied, fluorescence microscopy studies confirm that bacterial cells with exogenous cholesterol in the growth medium develop cholesterol-enriched lipid clusters in the cell membrane. This lipid clustering does not occur if cholesterol is added to the culture of a sphingolipid-negative mutant strain or in stress-free conditions. We have also identified a candidate sphingolipid signaling molecule, as well as possible genes that can carry out cholesterol uptake and sphingolipid-signal production. We hypothesize that Bacteroides spp. use sphingolipids/cholesterol-dependent signaling pathway to withstand various stresses and establish itself in the potentially hostile intestinal environment. In this study, our specific aims are to determine 1) the signaling pathway by defining the key components in the circuitry and 2) whether the signaling is important for maintaining Bacteroides as a major bacterial component in the mammalian GI tract.